The long-term goal of our research is to understand
the function of the nicotinic acetylcholine (ACh) receptors in terms
of their molecular structures (Karlin, 2002). As we originally showed,
the muscle-type ACh receptors have five subunits arranged around
the central channel in the order alpha-gamma-alpha-beta-delta (Karlin
et al., 1983) (Figure 1).

Each subunit has four membrane spanning segments
(M1-M4), and our recent research has been aimed at the structure
and function of these segments. We are attempting to classify every
residue in M1–M4 as water facing, lipid facing, or buried.
Many of the water-facing residues, which include the residues lining
of the channel, have been identified by application of the substituted
cysteine-accessibility method (SCAM), which we developed (Karlin
and Akabas, 1998) (Figure 2).

In SCAM, reactions are quantitated by their perturbation
of receptor function, as measured electrophysiologically. Using
this approach, we have also mapped the resting (Wilson and Karlin,
1998) and desensitization (Wilson and Karlin, 2001) gates (Figure
3), the intrinsic electrostatic potential in the open and resting
channel (Pascual and Karlin, 1998; Wilson et al., 2000) (Figure
4) and the site of binding of open-channel blockers (Pascual and
Karlin, 1998; Yu, Shi, and Karlin, 2003) (Figure 5).

Using a different approach that we developed on
a KcsA, a bacterial potassium channel (Li, Shi, and Karlin, 2003),
we are identifying the lipid facing residues, substituted by cysteines,
by their photoreaction, in the presence and absence of ACh, with
a hydrophobic photolabel that strongly prefers cysteine, quantitating
the reactivity of residues by a cysteine specific gel shift assay.
In addition we are mapping the relative arrangement and paths through
the membrane of M1-M4 by crosslinking. The surface exposure of each
residue and the paths of the segments through the membrane will
be the bases for a detailed model of the 2°, 3° and 4°
structure of the membrane domain. The differences in reaction rates
in the presence and in the absence of ACh will identify structural
elements that move during changes in the functional state of the
receptor.